Neurodegenerative diseases and CNS lesions such as spinal cord and brain injuries are widespread conditions with devastating effects on the life of the patients and their close relatives. As an example, about 450,000 people in the USA live with spinal cord injury (1 in 670) and only 5% of patients with complete injury recover locomotion. Almost more dramatically, about 2,000,000 US people undergo each year a traumatic brain injury (TBI), whose immediate as well as long term consequences are largely underestimated and deprived of medical solution, TBI provokes traumatic lesions of the brain that occur through a direct or indirect physical impact, either focal or diffuse like a blast an explosion. Long term consequences of TBI include mood disorders, sleep disorders, cognitive memory loss, locomotor disabilities and occur very frequently unrespectively of the initial severity of the trauma, racing an increasing number of helpless brain post-traumatic situations in soldiers back from battle field, the US government recently declared traumatic brain injury as a real epidemy and a major orphan sector.
At the biological level, TBI provokes widespread shearing and stretching of nerve fibers (diffuse axonal injury) and leads to destabilization of neuronal microtubules, disruption of the cytoskeleton, dendrite atrophy and loss of MAPs. These damages are important contributors to the functional impairments after TBI because microtubules, neurofilaments and microfilaments of the neuronal cytoskeleton are essential for the physiological functions of the neuronal cell. The functional consequences of dendritic damage are reflected in reports of compromised efficacy of synaptic transmission following TBI. After TBI, a spontaneous and partial recovery of lost function can occur over time although axonal regeneration is extremely limited in the mammalian adult central nervous system. The underlying mechanisms of this recovery are not fully understood but they involve the reorganization of connectivity with the formation of new synapses between neurons (Thompson et al., 2006). These observations have important therapeutic implications in humans, and were part of the inventor's strategy to develop therapies that stimulate plasticity to maximize the recovery of function.
Despite many research efforts in the field, effective molecules for the treatment of neurodegenerative diseases and CNS lesions are still not available. Interestingly, neurodegenerative diseases and CNS lesions share many pathogenetical similarities including deterioration of neuronal cytoskeleton. This deterioration can be the consequence but also the cause of damage to the affected cells. Growing evidence suggest that the cytoskeletal degradation observed after spinal cord injury (Zhang et al., J. Neuropathol Exp Neurol 2000) results from increased extracellular glutamate which in turn increases intracellular Ca++ activation of ion channels. Thus, the accumulation of intracellular Ca++ (i.e. Ca++ overload) can activate the protease calpain which induces proteolysis of MAP2 and TAU leading to abnormal microtubule depolymerization. The use of a calpain inhibitor (Schumacher et al., J Neurochem 2000) and the salting-out of glutamate (Springer et al., J Neurochem 2997) decrease the consequences of spinal cord injury in rodents by partially preserving the cytoskeleton.
Mood disorders include major and bipolar depression and are common, chronic and life threatening illnesses in Western society (Maris, Lancet 2002). Major depression affects 8-12% of the population (Andrade et al., Int. J. Meth. Psychiatr. Res. 2003) and 15% of suicides are committed by depressives in the USA (Manji et al., Nat. Med. 2001). Major depression is traditionally associated with low levels of the central nervous system (CNS) monoamines (i.e. serotonin [5-HT], dopamine [DA], norepinephrine [NE]). Antidepressant drugs target monoaminergic function by preventing their reuptake presynaptically cc blocking their metabolism. Although antidepressants seem to exert their initial effect by immediately increasing monoaminergic levels intrasynaptically (Malaglè et al., Eur. J. Pharmacol. 1995; Romero et al., J. Neurochem. 1996) their clinical efficacy occurs only after chronic administration (Blier and de Montigny, TiPS 1994). These findings lead the scientific community to the novel hypothesis that enhanced monoaminergic neurotransmission per se is not sufficient to explain the clinical actions of antidepressant drugs (Warner-Schmidt and Duman, Hippocampus 2006). Recently, magnetic resonance studies showed volume loss and structural abnormality in the hippocampus of depressed individuals (Campbell and Macqueen, J. Psychiatry Neurosci, 2004). Stress and major depression appear to be closely related and pre-clinical studies employing stress as predisposing factor to depression suggest that the hippocampal structural alterations observed in depressed patients may result from dendritic atrophy, neuriteal alterations, structural glial changes and neurogenesis decrease (Warner-Schmidt and Duman, Hippocampus 2006). Importantly, chronic treatment with antidepressant drugs seems to prevent stress-induced neuronal plasticity alterations in rodents (Warner-Schmidt and Duman, Hippocampus 2006). Consequently, the pathogenesis of depression may also involve stress-induced structural alterations in specific brain regions with the same features of dendrite alterations and impaired neurogenesis. The dynamics of the cytoskeletal microtubular system are fundamental for the formation and maintenance of synaptic connectivity including remodeling and extension of neurites (Mitchison and Kirschner, Neuron 1988) and dendrites (Valliant at al, neuron 2002). Microtubules are formed by the polymerization of the tubulin α/β heterodimers and in higher vertebrates three α-tubulins (α1, α2, and α4) and five β-tubulin (βI, βII, βIII, βIVα and βIVb) isotypes are specifically expressed in the brain (Luduena, Int. Rev. Cytol. 1998). Microtubules specifically interact with different proteins named microtubule-associated proteins (MAPs), between them the microtubule-associated protein 2 (MAP2) isoforms and TAO represent the major components of the proteins interacting with neuronal microtubules. They are present in all the extensions which constitute the dendritic arborization of a neuron, such neuronal branches are a key factor for the establishment of synaptic connections (Matus, Microtubules 1994; Sanchez et al., Prog. Neurobiol 2000). MAP2 proteins are necessary for the formation of dendrites since suppression of MAP2 synthesis caused either neuritic growth to stop in neurons in culture (Caceres et al. Neuron 1992) or dendritic growth to stop in MAP2 knockout mice (Harada et al., J. Cell. Biol. 2002). However, the synthesis of MAP2 proteins is not in and of itself sufficient to induce this dendritic growth process. Certain steroids such as estradiol or progesterone can induce an increase in MAP2 synthesis without . . . inducing significant morphological changes (Reyna-Neyra at al., Brain Res. 2002). Recent data showed that experimental models of stress and depression such as restraint stress (Bianchi et al. Synapse 2003), forced swimming test (Bianchi at al., Curr. Drug Targets CNS Neurol. Disord. 2005) and social isolation (Bianchi et al., EJN 2006) induce abnormal microtubule stabilization and dendrite retraction in rat hippocampus. Additionally, different stressors and administration of glucocorticoids can change the expression of different MAPs including MAP-2, MAP-1A and TAU in rat hippocampus (for an extensive review see Bianchi et al., Curr. Drug Targets CNS Neurol. Disord. 2005).
Finally, antidepressant drugs can affect MAP2 function and in turn the dynamics of the microtubular system. Indeed, both 5-HT and NP reuptake inhibitors differentially increased MAP2 phosphorylation and decreased microtubule assembly (i.e. increased microtubule dynamics) in rat cerebral cortex (Perez at al., Neuropsychopharmacology 1991; Miyamoto et al., Eur. J. Pharmacol., 1997) and in neuroblastoma cells (Donati and Rasenick, Neuropsychopharmacology 2005). Taken all together, these findings lead to the original hypothesis that the pathogenesis and treatment of depression may include changes in microtubule dynamics (Bianchi et al., Curr. Drug Targets CNS Neurol. Disord. 2005).
Neurodegenerative disorders, traumatic brain and spinal cord injuries and depressive disorders thus share alterations in microtubules dynamics and MAP2 expression and function-Importantly, the MAP2/TAU loss and microtubule depolymerization observed in spinal cord and brain injury can be directly responsible for the dysfunction of certain neurons and can result in their death. Moreover, such cytoskeletal deterioration can affect the number and the length of the dendritic extensions of the remaining neuronal cells and, as a consequence, decreases their effectiveness. Consistently, treatment with nerve growth factor (NGF), which prevents dendritic atrophy, enables better functional recovery after a lesion of the cerebral cortex in the rat (Kolb et al., Neuroscience 1997).
The existence of stem cells in certain regions of the central nervous system is well established today. Lesions stimulate the proliferation of these cells. However, these cells must migrate and differentiate. Differentiation implies, at a fundamental level, the development of the cytoskeleton.
It has been shown recently that, after cerebral ischemia, stem cells could differentiate into neurons and become integrated into the existing neuronal circuits (Nakatomi et al, Cell 2002). Similarly, it is well established that antidepressant drugs stimulate neurogenesis in the sub-granular zone of adult hippocampus and newborn cell migrate to the granule cell layer to become mature neurons extending dendrites and neurites (Warner-Schmidt and Duman, Hippocampus 2006).
The stimulation of dendrite and/or neurite growth (neuronal branching outgrowth) in these stem cells, and in already existing mature neurons, by molecules that improve tubulin polymerization and microtubule function could increase and or recover the number of functional synaptic connections.
Pregnenolone (PREG) binds to MAP2 and has the extremely important and original property of reinforcing the activity of this protein, namely its role in the activation of the tubulin polymerization process and the establishment of microtubular structures of greater function (Murakami et al., Proc Natl Acad Sci USA 2000).
In spite of much research, at present no specific targets other than MAPs have been identified for PREG.
MAP2 protein is found primarily in neurons. It is therefore probable that MAP2 binding molecules mainly target the cells of the nervous system, without having a notable action on other cellular types in which the concentration of MAP2 is very low.
Studies that demonstrate an in vivo effect by PREG are very few but they suggest a beneficial role for this steroid. It was shown that PREG administration decreased the reaction (formation of gliotic tissue (i.e. accumulation of astrocytes)) following a penetrating lesion in rat cerebral cortex and hippocampus (Garcia-Estrada et al, Int. J Devl Neuroscience 1999). Additionally, PREG administration reverses the age-dependent accumulation of glial fibrillary acidic protein within astrocytes of specific regions of the rat brain (Legrand and Alonso, Brain Res. 1998), PREG also contributed to improved functional recovery after a spinal cord trauma (Guth et al., Proc Natl Acad Sci USA 1994). Furthermore, PREG was showed to protect against toxicity induced by glutamate and the protein beta amyloid in hippocampal cells line (HT-22) cultures (Gursoy et al., Neurochem Res. 2001).
On the other hand, decreased levels of PREG have been reported in the cerebrospinal fluid of depressed patients (George et al., Biol. Psych. 1994). Furthermore, a first clinical investigation on the effects of PREG in healthy volunteers revealed a general tendency of reduced subjective depression ratings (Meieran et al., Psychoneuroendocrinology 2004). Moreover, antidepressant drugs increased PREG levels in rat hippocampus (Serra et al., Psychopharmacology 2001), while models of depression such as social isolation decreased it (Serra et al., J. Neurochem. 2000). Thus, PREG has been shown as having some beneficial effect in the CNS, both in case of traumatic lesion and in case of depressive disorders.
PREG is a metabolite of cholesterol and the precursor of all steroid hormones. The synthesis of these hormones implies the conversion of the PREG Δ5-3β-OH structure to Δ4-3-ketone derivatives (implemented by an enzyme called 3βHED). The Applicant blocked the Δ5-3β-OH structure to prevent this metabolic conversion and also to prevent, the formation of the ester sulfate, of PREG, a molecule that can be detrimental at high concentrations. Thus, the Applicant has revealed a compound, 3-methoxy-pregnenolone (3β-methoxy-pregna-5-ene-20-one, abbreviated as 3-methoxy-PREG), which possesses these properties and which, moreover, is at least as active as PREG in promoting microtubule polymerization and function. It was verified by mass spectrometry coupled with gas chromatography that 3-methoxy-PREG is not converted into PREG.
3-methoxy-PREG is also not converted into PREG metabolites, such as progesterone (PROG) and its further metabolites with progestative activity anon as allopregnanolone or epipregnanolone or pregnenolone sulfate. As a result, 3-methoxy-PREG has no progestative activity and is thus not a progestin. It was further verified that 3-methoxy-PREG does not have any agonist activity on many steroid hormone receptors (see Examples 11 and 13). 3-methoxy-PREG thus has no steroid hormone activity, which may further limit potential adverse effects in vivo.